Chloromethane (CH3Cl, methyl chloride) is the most abundant volatile halocarbon in the atmosphere and involved in stratospheric ozone depletion. The global CH3Cl budget, and especially the CH3Cl sink from microbial degradation in soil, still involves large uncertainties. These may potentially be resolved by a combination of stable isotope analysis and bacterial diversity studies. We determined the stable isotope fractionation of CH3Cl hydrogen and carbon and investigated bacterial diversity during CH3Cl degradation in three soils with different properties (forest, grassland, and agricultural soils) and at different temperatures and headspace mixing ratios of CH3Cl. The extent of chloromethane degradation decreased in the order forest > grassland > agricultural soil. Rates ranged from 0.7 to 2.5 μg g−1 dry wt. d−1 for forest soil, from 0.1 to 0.9 μg g−1 dry wt. d−1 for grassland soil, and from 0.1 to 0.4 μg g−1 dry wt. d−1 for agricultural soil and increased with increasing temperature and CH3Cl supplementation. The measured mean stable hydrogen enrichment factor of CH3Cl of −50 ± 13‰ was unaffected by temperature, mixing ratio, or soil type. In contrast, the stable carbon enrichment factor depended on CH3Cl degradation rates and ranged from −38 to −11‰. Bacterial community composition correlated with soil properties was independent from CH3Cl degradation or isotope enrichment. Nevertheless, increased abundance after CH3Cl incubation was observed in 21 bacterial operational taxonomical units (OTUs at the 97% 16S RNA sequence identity level). This suggests that some of these bacterial taxa, although not previously associated with CH3Cl degradation, may play a role in the microbial CH3Cl sink in soil.